The research proposed in Project 4 will strive to elucidate the mechanisms by which general anesthetics modulate ion channels in the central nervous system to induce reversible immobilization and amnesia. Sensitivity to inhibition, potentiation, or activation by anesthetics is observed in several ion channel families; ligand-gated ion channels (LGIC), and voltage-gated cation channels (VGCCs), which may all serve to transduce the clinical effects of general anesthetics. Project 4 will test the hypothesis that modulation by anesthetics reflects a combination pore and allosteric sites, and that it is the balance of affinities that cor\trol the ultimate effects of anesthetics on electrophysiology. Aim 1 will rely on the unique capacity of computer simulations based on fully atomistic models of the relevant membrane proteins in a hydrated membrane environment to probe structural details and measure affinities of anesthetics for isolated binding sites. To this end, we will measure the binding affinities of isoflurane and propofol to both the channel lumen and two previously identified transmembrane sites on the nicotinic acetylcholine receptor (nAChR) and yaminobutyric acid class A receptor (GABAAr) ofthe Cys-loop superfamily using the alchemical free energy perturbation (FEP) methodology. Additional sites identified from photoaifinity labeling to Cys-loop receptors in Project 1 (Eckenhoff) will also be included in this Aim, resulting in a ranking for likely significance of each site. In Aim 2 we will employ computer simulations to gain insight into determinants of sensitivity in VGCCs. In detail, we will identify binding sites and compute their affinities of isoflurane and propofol for anestheticsensitive K-Shaw2 and the prokaryotic NaChBac. Our computations will also aid in interpretation of results of Project 2 (Covanrubias), by focusing on the role of the possible allosteric site (S4-S5 linker) in these channels. Specific Aim 3 will build upon a recently derived molecular dynamics (MD) simulation model for the NaChBac to explore the effects of anesthetics on the various functional states of the channel (e.g., open, closed, resting etc). In detail, we will expose membrane-bound models of NaChBac to clinical concentrations of isoflurane, and propofol (and other interesting anesthetics developed in Project 1) in long-time MD simulations to determine plausible binding sites and thereby link to experimental activities in other projects. The project will also develop a model for sevofluorane.